Abstract
Biofilm-associated infections with Staphylococcus aureus are difficult to treat even after administration of antibiotics that according to the standard susceptibility assays are effective. Currently, the assays used in the clinical laboratories to determine the sensitivity of S. aureus towards antibiotics are not representing the behaviour of biofilm-associated S. aureus, since these assays are performed on planktonic bacteria. In research settings, microcalorimetry has been used for antibiotic susceptibility studies. Therefore, in this study we investigated if we can use isothermal microcalorimetry to monitor the response of biofilm towards antibiotic treatment in real-time. We developed a reproducible method to generate biofilm in an isothermal microcalorimeter setup. Using this system, the sensitivity of 5 methicillin-sensitive S. aureus (MSSA) and 5 methicillin-resistant S. aureus (MRSA) strains from different genetic lineages were determined towards: flucloxacillin, cefuroxime, cefotaxime, gentamicin, rifampicin, vancomycin, levofloxacin, clindamycin, erythromycin, linezolid, fusidic acid, co-trimoxazole, and doxycycline. In contrast to conventional assays, our calorimetry-based biofilm susceptibility assay showed that S. aureus biofilms, regardless MSSA or MRSA, can survive the exposure to the maximum serum concentration of all tested antibiotics. The only treatment with a single antibiotic showing a significant reduction in biofilm survival was rifampicin, yet in 20% of the strains, emerging antibiotic resistance was observed. Furthermore, the combination of rifampicin with flucloxacillin, vancomycin or levofloxacin was able to prevent S. aureus biofilm from becoming resistant to rifampicin. Isothermal microcalorimetry allows real-time monitoring of the sensitivity of S. aureus biofilms towards antibiotics in a fast and reliable way.
Highlights
Staphylococcus aureus is a notorious pathogen in post-surgery complications and severe infections as endocarditis, bacteremia and bone and joint infections [1, 2]
To determine the minimal inhibitory concentration (MIC) of flucloxacillin, cefuroxime, cefotaxime, gentamicin, rifampicin, clindamycin, erythromycin, vancomycin, linezolid, levofloxacin, fusidic acid, co-trimoxazole, and doxycycline, broth microdilution (BMD) assay was performed on all strains according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST)
antimicrobial susceptibility testing (AST) results from VITEK1 2 system showed that S. aureus CC5 Mu50 was resistant to almost all of the 13 tested antibiotics, except for linezolid, fusidic acid, and co-trimoxazole (Table 3), it was used as non-susceptible reference strain
Summary
Staphylococcus aureus is a notorious pathogen in post-surgery complications and severe infections as endocarditis, bacteremia and bone and joint infections [1, 2]. The EPS matrix as a shield makes antibiotic therapy more difficult, leading to prolonged infections and more severe complications including bacteremia and death [22,23,24,25]. Despite the fact that bacteria from biofilms are different from their planktonic counterparts, the current antimicrobial susceptibility testing (AST) for S. aureus isolated from biofilm-related infections still uses planktonic bacteria. This practice leads to overestimation of antibiotic effectivity as biofilm-associated bacteria show an increase tolerance towards antibiotics [26, 27]. The development of persister cells [29,30,31] and extracellular matrix formation [14,15,16,17,18,19,20] are among to the main causes of biofilm tolerance towards many antibiotic treatments
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